Throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure in their entirety.
Also, throughout this application various nonstandard abbreviations are used as follows: H2 relaxin (relaxin-2); ΔH2 (mutated/modified H2 relaxin); rH2 (recombinant H2 relaxin); RBD (receptor binding domain); LRR (leucine rich region); LV (lentiviral vector); UHN (University Health Network); eGFP (enhanced GFP); PMH (Princess Margaret Hospital); BLI (bioluminescence imaging); and CM (conditioned medium).
Humans possess three distinct genes for relaxin, termed relaxin-1 (H1), relaxin 2 (H2) and relaxin-3 (H3). All insulin and relaxin-like peptides share a homologous molecular structure that define them as members of the insulin-like superfamily. Each mature peptide (˜6-7 kDa) consists of an A and B chain joined by two interchain disulfide bonds, and one intrachain disulfide bond contained in the A chain (Sherwood, O D. (2004). Relaxins physiological roles and other diverse actions. Endocr Rev. 25:205-34).
Relaxin is classically known as a reproductive hormone with peak levels circulating during pregnancy (Sherwood, O D. (2004). Relaxins physiological roles and other diverse actions. Endocr Rev. 25:205-34). However, studies have broadened the scope for relaxin biology and it is now considered a pleiotropic hormone with functions in the cardiovascular and central nervous systems, neovascularization, and as an agent involved in ECM and connective tissue remodeling believed to be mediated by matrix metalloproteinases (MMPs) (Bani, D. 1997. Relaxin: a plelotropic hormone. Gen Pharmacol. 28:13-22; Bathgate, R. A., Samuel, C. S., Burazin, T. C., Gundlach, A. L., Tregear, G. W. 2003. Relaxin: new peptides, receptors and novel actions. Trends Endocrinol Metab. 14:207-1 3). Due to its several functions, relaxin has been the focus of research for a variety of uses. PCT/US01/42484 describes methods of modulating apoptosis by the use of relaxin agonists or antagonists. PCT/AU2004/000798 describes cyclic peptide analogues of the relaxin superfamily proteins where such peptides may be used for treating hyperplastic or neoplastic disorders. Further, the overexpression of H2 relaxin from PC-3 prostate xenograft tumors has been demonstrated to exhibit increased growth compared to controls (Silvertown, J. D., Ng, J., Sato, T., Summerlee, A. J., Medin, J. A. 2006. H2 relaxl; overexpression increases in vivo prostate xenograft tumor growth and angiogenesis. Iift J Cancer. 118:62-73).
Peptides of the relaxin family have a conserved receptor binding domain (RBD) located on the B-chain (Sherwood, O D. (2004). Relaxins physiological roles and other diverse actions. Endocr Rev. 25:205-34). All H1, H2, H3 hormones and their orthologues sequenced to date have the Arg-X-X X-Arg-X-X-Ile motif at positions B13, B17 and B20, with the exception of porcine relaxin (H2 equivalent in human) which has a valine residue at B20 (Sherwood, O D. (2004). Relaxins physiological roles and other diverse actions. Endocr Rev. 25:205 34). Studies have confirmed that this RBD motif is essential for relaxin biological activity and high-affinity receptor binding (Bullesbach, E. E., Schwabe, C. 2000. The relaxin receptor-binding site geometry suggests a novel gripping mode of interaction. J Biol. Chem. 275:35276-80; Bullesbach, E. E., Yang, S., Schwabe, C. 1992. The receptor-binding site of human relaxin H. A dual prong-binding mechanism. J Biol. Chem. 267:22957-60). H2 relaxin derivatives have been synthesized with the B13 and B17 arginines replaced with other amino acid residues (Bullesbach, E. E., Yang, S., Schwabe, C. 1992. The receptor-binding site of human relaxin II. A dual prong binding mechanism. J Biol. Chem. 267:22957-60). In particular, a dilysine H2 relaxin (at sites B13, B17) was synthesized that was rendered biologically inactive, however, it retained about a 2200-fold lower affinity for receptor binding than the level of wild type H2 relaxin. Further, studies demonstrated that substitution of the isoleucine residue at B20 with alanine reduced receptor binding by 3 orders of magnitude (Bullesbach, E. E., Schwabe, C. 2000. The relaxin receptor-binding site geometry suggests a novel gripping mode of interaction. J Biol. Chem. 275:35276-80).
To date, no effective relaxin peptide antagonists have been described in the literature. The LGR7 relaxin receptor has been exploited as a potential antagonist to neutralize relaxin activity. Soluble LGR7 ectodomains were demonstrated to bind relaxin and inhibit receptor mediated signaling of cAMP and suppress nipple development in mice (Hsu, S. Y., Nakabayashi, K., Nishi, S., Kumagai, J., Kudo, M., Sherwood, O D., Hsueh, A. J. 2002. Activation of orphan receptors by the hormone relaxin. Science. 295:671-74). Additionally, a truncated LGR7 splice variant was reported to abrogate relaxin LGR7 binding in vitro (Scott D. J., Tregear, G. W., Bathgate, R A. 2005. LGR7 truncate is a splice variant of the relaxin receptor LGR7 and is a relaxin antagonist in vitro. Ann NY Acad Sci. 1041:22-C).
While studies have demonstrated the importance of the RBD sequence for relaxin binding, there has not previously been demonstrated any modified (mutant/altered) H2 relaxin that binds to the receptor with high affinity and effectively acts as an antagonist of the receptor. Also, a modified relaxin H2 antagonist has never previously been developed that binds to the receptor and is involved in tumor suppression. Lastly, the ΔH2 relaxin antagonist described in the present invention essentially blocks relaxin signaling the receptor presented on the cell surface instead of neutralizing circulating hormone.